Hydatid cysts strains identification by mitochondrial dehydrogenase NADH subunit5 isolated from cattle and buffalo host in Babylon governorate, Iraq

1. Hydatid cysts in cattle and buffalo in Babylon governorate.
2. Detection of genotypes of hydatid cysts.
3. Use of molecular methods in the diagnosis of Cestoda.

Introduction

The most dangerous tapeworm infection that affects human health is Echinococcus, which causes hydatid disease and is caused by E. granulosus larvae (1,2). The disease is economically significant in terms of morbidity and mortality (3), especially in light of the discovery of several bacterial contaminations in hydatid cysts (4). Domestic animals are one of the many intermediate host species that the larvae of this worm can infect. The adaptation has made it available everywhere (5). This parasite has a complicated life cycle. The hydatid cysts HC are found in most organs of herbivores, such as liver, lung, spleen, brain, etc., while the mature worm is attached to the intestine of the Canidae family as a final host (6). The infection of HC leads to a disorder in the functions of the intermediate host organs (7). Consequently, it had a significant impact on the global economy (8). Genetic diversity may affect several different phenotypic characteristics in E. granulosus, such as host type, maturity rate, antigenicity, and spatial distribution (9). According to complete mitochondrial DNA sequencing, there are ten separate genotypes (G1- G10) of E. granulosus sensu lato (10). The most common strain is E. granulosus sensu stricto (G1-G3), found worldwide (11). Many other parasitological studies have been conducted in this governorate, such as studies of (12-16), but few on the E. granulosus parasite. While They implemented many local studies to detect the types and strains of hydatid cysts in other governorates, The usage of cox1 and nad1 was most common (17-23). The cox1 and nad1 are not insufficient to designate genotypes (6), while Kinkar et al. (24) refer to the nad5 gene region alone as giving enough data to distinguish between genotypes.

So, this study was designed to determine causative strain for hydatid cysts isolated from (the livers and lungs) of cows and buffaloes in Babylon governorate, based on primer (mt NAD5). A few studies used this gene in Iraq in general, and it was not used in a previous study in Babylon governorate, Iraq.

Materials and methods

Ethical approve

The approval was given to conduct this scientific work by the University of Thi-Qar, College of Science in their book No. 3/7/2854 on 6/3/2022.

Samples collection

Through the period from March to October 2022, collection of a total of 40 samples of hydatid cysts were collected. Obtained from Babylon governorate slaughterhouses as shown in (Table 1), the contents of hydatid cysts of chosen samples were centrifuged four times with a power of 300/ 10 minutes, each time using normal saline, and cyst fluid was examined under a microscope to determine the fertility based to presence protoscolices (25,26). After that, the sediments of the hydatid cyst were kept at freezing until the DNA extraction process (27).

Table 1: The number of infected Hydatid cysts is based on host, organ, and fertility

DNA extraction

 Following the instructions on the kit's purification, DNA was extracted from hydatid cysts and then frozen for later use.

Technique of polymerase chain reaction (PCR)

 Forward and reverse primers were employed: F5′-GCC CCI ACI CCA GTI AGT TCT-3′ R5′-AAI ACA CTT AGA IAC ICC ATG ACT-3′ with molecular weight 297 bp, which were designed by Roelfsema et al. (28). All samples of previously extracted DNA were amplified using the traditional PCR technique. The final PCR reaction volume was 25 μl, which was made up of 0.5 μl of forward and reverse primers, 14 μl of nuclease-free water, five μl Master mix, and five μl of DNA. The following steps were used to carry out the reaction in a thermocycler (Table 2). DNA samples were put into the suitable wells of the TAE agarose gel 1.5% (w/v) and were dyed with 2μl of ethidium bromide dye. For 90 minutes at 70V, the agarose gel was run. Using a UV transilluminator, the DNA was visualized within an agarose gel. The length of the product of mt nad5 was approximately 297 bp, and 100 bp was the size marker on the gels.

Table 2: PCR condition for NAD5 gene

DNA Sequencing.

For the purified PCR products obtained by the Geneaid DNA Cleanup kit, as described above, a tool named Bioedit sequence alignment editor v.7.2.5 was used to fix the genetic data. The samples' hydatid cyst sequences were added to the NCBI database and assigned accession numbers (https://www.ncbi.n.lmnih.gov/).

Phylogenetic tree

The Maximum Composite of Likelihood method used genetic distance, UPGMA, and MEGA-X software v.11 for Phylogenetic tree analysis (29).

Results

Mitochondrial NAD₅ Gene

Using mt nad5 primer, we examine all chosen samples (12 samples), successfully ~ 297 bp (Figure 1).

Figure 1: Agarose gel electrophoresis of PCR amplification NAD5. (L: 100bp DNA ladder, lanes 1-9: contain PCR products derived from: livers lungs of Cattle in lanes 1- 4, buffalo in lanes 5- 9 represent).

For all the chosen 12 isolates, the partial sequencing of mt nad5 produced a sequence of ~ 297 bp. The percentage of the sequence is identical between the study samples and other samples in NCBI-BLAST, ranging from 100%-95%. The sequences obtained have been deposited in the Genbank database under these accession numbers: LC775105, LC775106, LC775107, LC775108, LC775109, LC775110, LC775111, LC775112, LC775113, LC775114, LC775115, LC775116. Shows that G1 is the most prevalent and is followed by the G3 (Table 3).

Table 3: Genotypes of Echinococcus granulosus, its accession number, and identity ratio with other isolates for samples isolated from either livers and lungs Cattle and Buffalo based on the mt nad5 gene and NCBI- BLAST alignment tool

Phylogenetic analysis

Figure 2 showed evolutionary relationships between the mt nad5 sequences produced in this study and the sequences found in Genbank of the E. granulosus strains and also explained that samples of this study have genetic diversity compared to the reference strains in Genbank. Their sequences showed a correlation with local and regional sequences.

Figure 2: A Phylogenetic tree analysis based on mt nad5 gene partial sequence used for E. granulosus genotyping among this study's isolates and other isolations deposited in the GenBank database. The phylogenetic tree was constructed using the Unweight Pair Group method with Arithmetic Mean (UPGMA tree) in MEGA-X software v.11).

Discussion

Genotyping is the essential and first step for detection, controlling the risk of spreading E. granulosus parasite, thus decreasing the infection. The result of the current study, depending on gene sequence analysis mt nad5 297 bp, showed only two genotypes G1 and G3. The sheep strain G1 was the dominant 91.6%. Eleven samples were liver and lung cattle and buffalo, whereas only 8.6 % were buffalo G3, represented by one sample only, Accession No. LC775109.

The previous studies in different provinces of Iraq recorded the same result but have used all cox1, nad1, atp6, or one of them, such as study Hammad et al. (30) recorded and G3 strains only in Kirkuk and Sulaimania, also in Thi-Qar and Misan governorates (31-33). However, these results differed from the studies of Hamoo et al. (34), Abdulla et al. (35), were recorded G1 strain only in Aqrah and Koya cities in Dohuk and Erbil, northern Iraq, respectively, likewise recorded the same strain in other studies (36-38) from different governorates in middle Iraq and Kirkuk governorate respectively. Also disagreed with the studies of Al-Shimary (39), Al-Yaqoubi (40) in Al-Najaf and Al-Qadisiyah governorates, and Thi-Qar governorate respectively that recorded four genotypes together in addition to G1, G3, also recorded G2, G5. The results of this study may differ from previous studies due to different genetically contaminated ovules of study areas, or some studies use only the forward or reverse primer, and this produces an incomplete sequence for gene, compared with those used together so that the diagnosis may be inaccurate in these studies.

These results prove that G1 is the most infected strain of Echinococcus spp. in Iraq. This might be due to its large capacity to infect several herbivorous hosts (8). The strains recorded diverse haplotypes associated with the local and global strains, although the dominating G1 strain. This genetic diversity in the study area may result from the interaction between wildlife hosts and herbivores (41-44). Additionally, the definitive host might be infected by various genetically mature worms that reproduce by various fertilization as cross and self-fertilization, thus producing a variety of ovules; therefore, produced hydatid cysts are different genetically (45-47).

Conclusion

There is genetic diversity for haplotypes in G1 that recorded dominance strain, followed by G3 of larval E. granulosus (hydatid cysts) in Babylon province. Different genotypes of hydatid cysts may not have been the source of intermediate hosts. The mt NAD5 primer is reliable for identifying the parasite strains and can be used in future studies.

Acknowledgments

The author thanks the veterinarian employees at the Babylon governorate slaughterhouse, especially Dr. Aqeel Mohammed and Dr. Ahmed Ali, for helping us collect samples in this study.

Conflict of interest

The authors' interests do not conflict with this paper's publication.